European Journal of Medicinal Chemistry · 2018. 8. 15. · X-ray diffraction data collections were performed on an Enraf-Nonius Kappa-CCD diffractometer (95 mm CCD camera on k-goniostat)

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European Journal of Medicinal Chemistry 96 (2015) 491e503

Contents lists avai

European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Design, synthesis and structureeactivity relationship of phthalimidesendowed with dual antiproliferative and immunomodulatoryactivities

Marcos Veríssimo de Oliveira Cardoso a, *, Diogo Rodrigo Magalh~aes Moreira a,Gevanio Bezerra Oliveira Filho a, Suellen Melo Tiburcio Cavalcanti a,Lucas Cunha Duarte Coelho a, Jos�e Wanderlan Pontes Espíndola a, Laura Rubio Gonzalez a,Marcelo Montenegro Rabello a, Marcelo Zaldini Hernandes a,Paulo Michel Pinheiro Ferreira b, Cl�audia Pessoa c, d, Carlos Alberto de Simone e,Elisalva Teixeira Guimar~aes f, g, Milena Botelho Pereira Soares g, Ana Cristina Lima Leite a

a Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de Pernambuco, 50740-520, Recife, PE, Brazilb Departamento de Biofísica e Fisiologia, Programa de P�os-Graduaç~ao em Ciências Farmacêuticas, Universidade Federal do Piauí, 64049-550, Teresina, PI,Brazilc Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Cear�a, 60430-270, Fortaleza, CE, Brazild Fundaç~ao Oswaldo Cruz, 60180-900, Fortaleza, CE, Brasile Departamento de Física e Inform�atica, Instituto de Física, Universidade de S~ao Paulo, 13560-970, S~ao Carlos, SP, Brazilf Departamento de Ciências da Vida, Universidade do Estado da Bahia, 41150-000, Salvador, BA, Brazilg Hospital S~ao Rafael, 41253-190, Salvador, BA, Brazil

a r t i c l e i n f o

Article history:Received 11 November 2014Received in revised form15 April 2015Accepted 18 April 2015Available online 20 April 2015

Keywords:PhthalimideThiosemicarbazoneThiazoleThiazolidinoneAntiproliferativeImmunosuppressive

* Corresponding author.E-mail address: [email protected] (M.V

http://dx.doi.org/10.1016/j.ejmech.2015.04.0410223-5234/© 2015 Elsevier Masson SAS. All rights re

a b s t r a c t

The present work reports the synthesis and evaluation of the antitumour and immunomodulatoryproperties of new phthalimides derivatives designed to explore molecular hybridization and bio-isosterism approaches between thalidomide, thiosemicarbazone, thiazolidinone and thiazole series.Twenty-seven new molecules were assessed for their immunosuppressive effect toward TNFa, IFNg, IL-2and IL-6 production and antiproliferative activity. The best activity profile was observed for the (6aef)series, which presents phthalyl and thiazolidinone groups.

© 2015 Elsevier Masson SAS. All rights reserved.

1. Introduction

Thalidomide appears to be a multi-target drug that impinges ona number of seemingly distinct cellular processes, includingpeptidase inhibition, glucosidase inhibition, androgen receptorantagonism and (cyclooxygenase) COX inhibition [1].

One of the most studied biological activities influenced bythalidomide is the inhibition of the expression of the pro-

.O. Cardoso).

served.

inflammatory cytokine tumour necrosis factor (TNFa) [2]. TNF is acentral regulator of the inflammatory cascade that controls manyeffector pathways as anti-angiogenic, anti-inflammatory andimmunomodulatory molecule. The molecular mode of action ofthalidomide on TNFa expression is thought to involve the inflam-matory NFjB signalling pathway, specifically inhibiting the activityof the IjB kinase, IKKa [3].

Thalidomide is also known as an inhibitor of nuclear factorkappa B (NF-kB) activation [4e7]. NF-kB is a family of structurallyrelated transcription factors that play a major role in inflammationand immune responses. Moreover, NF-kB inhibits apoptosis, and

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Fig. 2. Design concept of target compounds.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503492

induces proliferation and angiogenesis, suggesting that NF-kB has apivotal role in oncogenesis and tumour progression [8,9].

Immunomodulatory drugs (IMiDs) are thalidomide derivativeswith improved anti-tumour activity and safer toxicity profiles [10].The two leading IMiD compounds, lenalidomide (CC-5013; IMiD3;Revlimid) and pomalidomide (CC-4047; IMiD1; Actimid), were thefirst drugs to enter into clinical trials for the treatment of multiplemyeloma in 1999 [11] and are the subject of clinical evaluation inother haematological malignancies [12]. Studies on the structur-eeactivity relationship (SAR) of the metabolites of thalidomide andits analogues have revealed that the phthalimide ring system is anessential pharmacophoric fragment [13].

In fact, substituted N-phenylphthalimides are of high interestbecause they have been found to inhibit TNFa [1,14] and COX [1],and have tubulin binding properties [15]. With these properties inmind, phthalimide has usually been employed in the design ofpotential antiinflammatory [16], immunomodulatory [17e19],antiangiogenic [20e22] and antitumour [23e26] drug candidates.In this promising scenario, the strategy of molecular hybridizationusing phthalimide as a pharmacophoric fragment have figuredprominently and led to many successful cases [14]. On the otherhand, thiosemicarbazones are compounds of considerable interestbecause of their important chemical properties and potentiallybeneficial biological activities [27e30].

In general, the synthesis of thiosemicarbazone compoundspresents low cost and high atom economy because all the atomsfrom the reagents (except the water liberated in the condensation)are present in the final molecule.

4-N-substituted thiosemicarbazones show remarkable activityin comparison with their unsubstituted counterparts. An enhancedinhibitory effect may be attributed to the increased lipophilicitythat allows the molecules to easily cross the cell membrane. The 4-N nitrogen of the thiosemicarbazone skeleton may contain: a) twohydrogen atoms (unsubstituted thiosemicarbazones); b) onehydrogen atom and one alkyl or aryl group and c) two alkyl or arylgroups or may be a part of a cyclic ring [31].

Bearing in mind the molecular pharmacophores outlined aboveand their structural requirements, some phthalimide derivativeswere designed after exploring molecular hybridization and bio-isosterism approaches between thalidomide, thiosemicarbazone,thiazolidinone and thiazole moieties (Fig. 1). These derivativeswere synthesized by our group and based on the obtained biolog-ical data, and new SAR information was collected. Furthermore, anumber of the derivatives exhibited potent in vivo activity againstS-180 sarcoma cells that was comparable to that of the referencedrug, thalidomide [32].

In a continuation of our work on the structureeactivity rela-tionship, twenty-seven new phthalimide derivatives were pre-pared to establish an appropriate SAR. Our designwas based on themolecular hybridization of the phthalimide ring system with athiosemicarbazone, thiazolidinone or thiazole subunit.

Fig. 1. Bioisosteric relationship between thalidomide and the pr

In the design concept, the 2-N and 4-N nitrogen of the thio-semicarbazone skeleton were then substituted by alkyl groups(2aec) to improve the lipophilicity. A set of compounds (3aed,4aef and 6aef) bearing thiazolidinones was then synthesized byexploring bioisosteric relationship between thiazolidinones andthiosemicarbazones. Our approach also investigated the homolo-gation between the phthalyl system and thiazolidinones (3aed and4aef) to investigate the influence of flexibility. Subsequently, abioisosteric exchange between the thiazolidinone and thiazolenuclei wasmade, so that the 4-N nitrogen of the thiosemicarbazoneskeleton was then converted to a thiazole ring that contained alkyl(7b) or phenyl groups (7a, 7ceh) (Fig. 2).

2. Results and discussion

2.1. Chemistry

The synthesis of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-aminederivatives was adapted from the method described previously[32,33] and is outlined in Scheme 1.

From the phthalic anhydride (1) obtained commercially, anacetal intermediate was first synthesized by imidification reactionwith aminoacetaldehyde diethyl acetal reagent in the presence ofDMAP. Then, this intermediate underwent acid hydrolysis to obtainthe aldehyde intermediate, which was condensed with

oposed thiosemicarbazones, thiazolidinones and thiazoles.

Scheme 1. Synthesis of target compounds. Reagents and conditions: a) aminoacetaldehyde diethyl acetal, toluene, DMAP, reflux, 2 h; H2O, H2SO4, reflux, 2 h; thiosemicarbazide,EtOH, H2SO4, reflux, 20 h b) BrCH2CO2CH3, AcONa, EtOH, reflux, 20 h; halogenated acids or esters, AcONa, EtOH, reflux, 20 h. c) W-ArCHO, AcOK, DMF, reflux, 12e20 h d) thio-semicarbazide, DMAP, DMF, reflux, 1 h; BrCH2CO2CH3, AcONa, EtOH, reflux, 24 h. e) W-ArCHO, AcOK, DMF, reflux, 8e24 h.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503 493

thiosemicarbazide for the synthesis of phthalyl thiosemicarbazones(2aec), producing crystals in good yields and in a short reactiontime. The thiazolidinone (3aed) was synthesized by the cyclizationof the thiosemicarbazones obtained with a-halogenated acids oresters in AcONa. Finally, through the reaction between the thiazo-lidinone (3a) and different aryl-aldehydes in basic medium thebenzylidyl-phthalyl-4-thiazolidinones (4aef) were obtained viaMichael reaction.

In addition to the benzylidyl-phthalyl-4-thiazolidinones, (6aef),a homologous series to the compounds (4aef), was also synthe-sized. This synthesis occurred primarily through the reaction be-tween the thiosemicarbazide and phthalic anhydride under acidicconditions, followed by cyclization of the thiosemicarbazone withmethyl bromoacetate to obtain the intermediate (5). Then, thisintermediate was treated with the same aryl-aldehydes used in thesynthesis of compounds (4aef).

The phthalyl-thiazoles (7aeh) were produced through a

Scheme 2. Synthesis of target compounds. Reagents and conditions: a)

cyclization reaction between the thiosemicarbazone (2a) anddifferent substituted 2-bromoacetophenone in NaOAc (Scheme 2).

The chemical structure of these products was established usingNMR (1H, 13C and DEPT), IR spectral and elemental analysis (for C, H,N, S).

2.2. X-ray analysis

X-ray diffraction data collections were performed on an Enraf-Nonius Kappa-CCD diffractometer (95 mm CCD camera on k-goniostat) using graphite monochromated MoKa radiation(0.71073 Å), at room temperature. Data collections were carried outusing the COLLECT software [34] up to 50� in 2q. The final unit cellparameters were based on 6064 reflections for the (2a) compoundand 3662 for the (2b) compound. Integration and scaling of thereflections, and correction for Lorentz and polarization effects wereperformed with the HKL DENZO-SCALEPACK system of programs

substituted 2-bromoacetophenone, AcONa, ethanol, reflux, 4e24 h.

Table 1Main crystallographic parameters of compounds (2a) and (2b).

Compound (2a) Compound (2b)

Empirical formula C11 H10 N4 O2 S H2O C12 H10 N4 O2 SFormula weight 280.3 274.3Crystal system monoclinic triclinicSpace group P21/c P-1a (Å) 5.4630(2) 10.1470(7)b (Å) 10.1120(4) 9.9790(5)c (Å) 23.9650(9) 14.0120(8)a (Å) 90.0 77.804(3)b (Å) 94.790(2) 87.552(3)g (Å) 90.0 67.989(3)V (Å3) 1319.25(2) 1284.67(2)Z 4 4Radiation (l, Å) MoKa (l ¼ 0.71070 Å) MoKa (l ¼ 0.71070 Å)m (mm�1) 0.255 0.255Absorption correction none noneTemp. (K) 295(2) 295(2)Dcalc (Mg m�3) 1.41 1.42Crystal dimensions (mm) 0.32 � 0.23 � 0.07 0.20 � 0.19 � 0.07q range (º) 3.3e27.5 3.1e27.3Reflections collected(Rint) 7398 [Rint ¼ 0.046] 13,033 [Rint ¼ 0.07]Independent reflections 2877 5040Data/parameters 2254/184 1880/343Goodness-of-fit on F2 1.038 0.937


[35]. The structures of compounds were solved by direct methodswith SHELXS-97 [36]. The models were refined by full-matrix leastsquares on F2 using SHELXL-97 [36]. The programORTEP-3 [37] wasused for graphic representation, and the program WINGX [38] wasused to prepare materials for publication. All H atoms were locatedby geometric considerations placed (CeH ¼ 0.93e0.97 Å;NeH ¼ 0.86 Å) and refined as riding with Uiso(H) ¼ 1.5Ueq(C-methyl) or 1.2Ueq(other). An Ortep-3 diagram of the molecules isshown in Fig. 3, and Table 1 shows the main crystallographic pa-rameters. All bond distances and angles, fractional coordinates,equivalent isotropic displacement parameters and other relevantinformation can be obtained free of charge from The CambridgeCrystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif under deposit numbers CCDC 972715 and CCDC972716, respectively.

Bond lengths and angles are in good agreement with the ex-pected values reported in the literature [39]. Compound (2a)crystallized with one solvent water molecule in the packing formOW-H1W/Si and OW-H2W/Sii [i ¼ xþ 1, y, z; ii ¼ -xþ 1, �y, �z],and hydrogen-bonding interactions where: H1W/Si ¼ 2.387(2)Å;OW-H1W/Si ¼ 169� and H2W/Sii ¼ 2.549(2) Å; OW-H2W/Sii ¼ 158�.

Final R indices (I > 2s (I)) R1 ¼ 0.041, wR2 ¼ 0.108 R1 ¼ 0.048,wR2 ¼ 0.124

R indices (all data) R1 ¼ 0.055, wR2 ¼ 0.121 R1 ¼ 0.134,wR2 ¼ 0.169

2.3. Pharmacological evaluation

Once their structures were elucidated, all compounds weretested as immunomodulatory and anticancer agents. First, the po-tential immunological properties of the compounds were assessedby measuring the secretion of cytokines from the animal macro-phages (TNFa and IL-6) and lymphocytes (IL-2 and IFNg). Thalid-omide (Thl) and dexamethasone (Dex) were used as controls.

TNFa, a highly pleiotropic cytokine produced primarily bymonocytes and macrophages, plays a central role in the host's

Fig. 3. ORTEP-3 projections of the compounds: (a) 2a and (b) 2b, showing the atom-numbering and displacement ellipsoids at the 50% probability level.

protective immune response to bacterial and viral infections[40,41]. However, it may also play a role in the pathogenesis ofdisease. Additionally, elevated levels of TNFa have been associatedwith fevers, malaise and weight loss that occur with chronic in-fections [42]. Otherwise, reductions in TNFa levels have been linkedwith an improvement in clinical symptoms in a number of diseasestates [43e45]. Immune stimulation with LPS was suitable foranalysing TNFa production, and it was observed that among the 27tested compounds, only four did not affect TNFa production at all(compounds (4bed) and (7e)). The inhibition profiles wereobserved for both concentrations tested (1 and 10 mg/mL); however,a better inhibition profile was observed at 10 mg/mL. Compounds(3a), (4a), (4e), (4f), (6eef), (7a) and (7d) showed average % inhi-bition cytokine between 52 and 73%. At the same concentration,thalidomide did not have an inhibitory profile (Fig. 4).

When observing the thiazolidinone group, series 6, showed bet-ter inhibition rates than did series 4, which contains a space groupbetween phthalyl and thiazolidinone ring. Phthalyl thio-semicarbazones (series 2) showed onlymoderate inhibitory activity.

IL-2 is instrumental in the body's natural response to microbialinfection and is normally produced by TH1 cells [46]. Levels of thiscytokine were significantly inhibited by compound (2c), (4a), (6a)and (6e) (45e72% range). We found that, among the twenty-sevensynthetic derivatives, compounds (4a) and (6e) displayed thestrongest ability to inhibit IL-2 secretion. Compounds (4bed), (4f),(6bed), (7d) and (7f) showed only a modest inhibition of IL-2(Fig. 5). It is worth mentioning that the inhibitory ability wasrevealed only at a concentration of 1 mg/mL.

For IL-2 cytokine, the thiazole nucleus (7aeh series) is inactivefor both concentrations. The activity was observed only at 1 mg/mL,and it seems that phthalyl thiazolidones at 1 mg/mL (series 4aefand 6aef) are selective for the inhibition of IL-2. Among the thia-zolidinone series, series 3aed was the only series that did notproduce inhibition. The main difference in this series is the ben-zylidine substituent at C5 of series (4aef). Compounds (4a) and(6e) were comparable to dexamethasone at the same concentra-tion. Once again, Thl was inactive at the same concentration.

http://www.ccdc.cam.ac.uk/data_request/cifhttp://www.ccdc.cam.ac.uk/data_request/cif

Fig. 4. Effect of phthalimides on TNF production. Peritoneal macrophages were incubated in the presence of phthalimides (1 and 10 mg) or medium alone, and stimulated or notwith bacterial lipopolysaccharide (LPS; 500 ng/mL). Thalidomide (Thl) and dexamethasone (Dex) were used as reference drugs in the same concentrations. Cell free supernatantswere collected 4 h later for cytokine analysis by sandwich ELISA (Duoset, R&D Systems kit). Data are the mean ± S.D. (error bars) obtained in duplicate; S.D., standard deviation.

Fig. 5. Effect of phthalimides on IL-2 production. Spleen cells were incubated in the presence of phthalimides (1 and 10 mg) or medium alone and were stimulated or not withconcanavalin A (ConA; 1 mg/mL). Thalidomide (Thl) and dexamethasone (Dex) were used as reference drugs at the same concentrations. Cell-free supernatants were collected 24 hlater for cytokine analysis by sandwich ELISA (Duoset, R&D Systems kit). Data are the mean ± S.D. (error bars) obtained in duplicate; S.D., standard deviation.


Interferon-g (IFNg) is a cytokine secreted by lymphocytes thatpromotes innate immunity, i.e., natural killer (NK) cells, and cellsthat are components of the adaptive immune system (specificsubsets of T cells) [47,48]. Furthermore, a role for IFNg in protec-tion against tumour development has recently been identified[49]. The results have shown that endogenously produced IFNg iscritical not only for the rejection of transplantable tumours butalso to prevent primary tumour development [50]. The level ofIFNg secretion was reduced by 95% (4b), 93% (7b), 87% (7d), 85%(7h), 83% (2a), and 82% (3b) at 1 mg/mL (Fig. 6). Derivative (4b) isthe strongest inhibitor of IFNg secretion among the twenty-sevencompounds and is comparable to dexamethasone (89%) and Thl

Fig. 6. Effect of phthalimides on IFNg secretion. Spleen cells were incubated in the preconcanavalin A (ConA; 1 mg/mL). Thalidomide (Thl) and dexamethasone (Dex) were used aslater for cytokine analysis by sandwich ELISA (Duoset, R&D Systems kit). Data are the mea

(93%). Both series representatives showed good inhibition activityat 1 mg/mL, but no trend can be identified with regard to spacinggroups or differences in ring structures (thiazolidinone versusthiazole).

IL-6, a pro-inflammatory cytokine, is secreted by the TH1 cellsand macrophages and stimulates the immune response to trauma,especially burns or other tissue damage leading to inflammation. Interms of the host response to a foreign pathogen, IL-6 has beenshown to provide resistance to mice against the bacterium, Strep-tococcus pneumoniae [51]. The effect of the tested compounds(10 mg/mL) on the secretion of this cytokine was only modest; theinhibition percentage did not reach 50%. Compounds (2c), (6aed),

sence of phthalimides (1 and 10 mg) or medium alone, and stimulated or not withreference drugs at the same concentrations. Cell-free supernatants were collected 24 hn ± S.D. (error bars) obtained in duplicate; S.D., standard deviation.

Fig. 7. Effect of phthalimides on IL-6 secretion. Peritoneal macrophages were incubated in the presence of phthalimides (1 and 10 mg) or medium alone, and stimulated or not withbacterial lipopolysaccharide (LPS; 500 ng/mL). Thalidomide (Thl) and dexamethasone (Dex) were used as reference drugs at the same concentrations. Cell-free supernatants werecollected 4 h later for cytokine analysis by sandwich ELISA (Duoset, R&D Systems kit). Data are the mean ± S.D. (error bars) obtained in duplicate; S.D., standard deviation.


(7a), (7c), (7d) and (7f) showed inhibition in the range of 31e39%(Fig. 7).

To investigate the anticancer properties of these compounds,these phthalimides were first evaluated against three tumour lines:MDA/MB-435 (melanoma), HCT-8 (colon) and SF-295 (nervoussystem).

Table 2 summarizes the cytotoxic action on tumour cells eval-uated by MTT assay. Compounds from series (6aef) were the mostpotent, especially compounds (6b) and (6f), which revealed cellproliferation inhibition rates ranging from 87.0 ± 11.1% (SF-295) to100 ± 1.1% (MDA/MB-435) and IC50 values of 7.5 and 5.3 mg/mL (SF-295) and 5.8 and 5.2 mg/mL (HCT-8), respectively. Doxorubicin, used

Table 2Screening of the in vitro cytotoxicity of 27 phthalimides derivatives on cancer cells atconcentration of 50 mg/mL and lymphocytes at 5 mg/mL. The cytotoxicity on bothneoplasic and normal cells was determined by MTT assay.

Sample SF-295 HCT-8 MDA/MB-435 LYMP (GI%) SD

2a NT 30.1 ± 0.8 17.8 ± 4.1 67.5% 2.9%2b NT 21.8 ± 6.1 28.5 ± 10.2 13.2% 4.4%2c NT 26.4 ± 0.2 22.0 ± 6.1 13.6% 4.9%3a NT 33.0 ± 13.0 8.0 ± 1.2 9.4% 8.8%3b 7.2 ± 6.4 26 ± 0.1 NT 2.4% 8.1%3c NT NT 5.8 ± 1.2 14.6% 16.1%3d NT NT 2.5 ± 0.3 35.7% 4.9%4a 23.4 ± 8.3 33.3 ± 6.5 24.9 ± 3.2 20.7% 1.9%4b 32.7 ± 2.0 33.7 ± 0.4 27.3 ± 7.5 NT 9.1%4c 56.2 ± 0.1 50.2 ± 13.1 34.4 ± 2.3 24.9% 3.3%4d 46.6 ± 9.5 47.5 ± 0.3 45.9 ± 14.0 16.7% 10.2%4e 16.7 ± 0.3 42.7 ± 2.0 11.2 ± 4.0 50.2% 42.3%4f 23.7 ± 9.5 42.2 ± 15.1 93.5 ± 1.0 32.1% 4.0%6a 67.1 ± 5.1 65.3 ± 2.7 30.2 ± 3.3 5.0% 12.4%6b 87.0 ± 11.1 97.1 ± 3.2 100.0 ± 1.1 1.4% 6.0%6c 32.1 ± 0.6 41.0 ± 0.1 50.7 ± 0.8 0.8% 2.6%6d 58.9 ± 2.3 56.5 ± 5.5 55.1 ± 5.7 NT 11.4%6e 62.5 ± 6.1 62.8 ± 7.9 60.4 ± 6.3 19.2% 10.7%6f 96.8 ± 0.3 91.3 ± 4.4 82.1 ± 8. 35.6% 23.8%7a NT 25.3 ± 9.8 35.5 ± 7.8 13.9% 3.4%7b 64.8 ± 1.0 45.8 ± 7.8 28.4 ± 3.0 59.9% 7.8%7c NT NT 24.8 ± 3.3 14.6% 14.2%7d 38.5 ± 1.2 52.3 ± 1.5 22.8 ± 1.9 4.0% 1.7%7e 7.5 ± 0.9 9.3 ± 3.7 16.3 ± 13.5 1.7% 4.4%7f NT NT NT 7.9% 2.8%7g 58.7 ± 3.5 72.5 ± 3.9 58.5 ± 1.8 9.8% 11.4%7h 11.1 ± 4.0 24.1 ± 1.1 NT 17.0% 23.2%Dox 100.00 ± 0.7 83.62 ± 2.9 96.7 ± 4.5 67.5% 2.9%Thl 10.4 ± 4.7 35.7 ± 3.2 40.5 ± 7.9 13.2% 4.4%

Data are presented as inhibition perceptual of the antiproliferative rate obtainedfrom at least three independent experiments for human tumor lines (SF-295, ner-vous system; HCT-8, colon; MDA/MB-435, melanoma) and normal human lym-phocytes (LYMP). Doxorubicin was used as positive control (Dox); Thalidomide asdrug of reference (Thl). NT: non toxic.

as positive control, was active against all lines. On the other hand,Thalidomide, the phthalimide of reference, wasweakly cytotoxic ontumour cells.

Likewise, the IC50 values for compounds (6b) and (6f) on humanlymphocytes were 9.4 and 7.7 mg/mL, respectively. As mentionedbefore, the selectivity between normal andmalignant cells is one ofthe critical issues for the research and development of chemo-therapeutic reagents.

In light of these findings, it is reasonable to draw some com-ments about the dual behaviour of compounds (6b) and (6f). Thesecompounds showed immunosuppressive activity toward TNFa at10 mg/mL and also showed anticancer properties against threetumour cell lines.

With regard to the structural features of the compounds and theimmunological profiles of all series of the tested compounds(Fig. 8), the immunosuppressive and antiproliferative profile of the(6aef) series of phthalimide derivatives were the most effective.The main structural difference between the (6aef) and (4aef) se-ries concerns the insertion of a flexible group (eCH2eCH]Ne)between the phthalyl and thiazolidinone rings (in the 4aef series).Another remark is the fact that series (7aeh) possesses a 4-phenyl-thiazole nucleus instead of a thiazolidin-4-one nucleus such as thatpresent in the (4aef) and (6aef) series.

It is worth mentioning that our previous results showed thatphthalimide derivatives were inactive (IC50 > 300 mM) in in vitrotests against four tumour cells lines: MDA/MB-435 (breast), HCT-8(colon), SF-295 (glioblastoma) and HL-60 (leukaemia). Likewise, ingeneral, these derivatives did not show immunosuppressiveproperties, which is a characteristic that is highly desirable in newimmunomodulatory drug candidates [32].

2.4. Docking studies

NF-kB is a significant transcription factor that regulates theexpression of various pro-survival genes. Themulti-subunit proteinkinase, IKK, regulates NF-kB activation in response to specificexternal mediators, including tumour necrosis factor-a (TNFa) andinterleukin-1 (IL-1) [52]. In the nucleus, NF-kB binds to its cognateDNA site and enhances the expression of a number of genes relatedto the immune response, cell proliferation and survival [53].

Thus, the inhibition of IKKb on the NF-kB pathway could beinvolved the anti-inflammatory and anti-cancer mechanism of themolecules reported in this work.

To understand a possible correlation between cell proliferationinhibition and IKKb, we investigated the interaction of compoundsreported in this work with IKKb (PDB ID: 4KIK) by conductingdocking studies. The binding mode for these ligands was

Fig. 8. Effect of substitutions in the thiazole ring on antiproliferative activity.


determined by the highest (most positive) score among thepossible solutions for each ligand. These calculations were gener-ated according to the ChemPLP Fitness Score [54].

Fig. 9 shows the trend observed between the in silico dockingscores and the cytotoxic activity on tumour cells evaluated by MTTassay. The assay indicates that the compounds with higher cyto-toxic action on tumour cells are usually those with the higherdocking scores, i.e., the molecules with high cytotoxic action alsohave a high affinity for the IKKb target, as revealed by the dockingscore values. It is important to highlight the large variation ofdocking scores (range: from 61.22 to 81.04), and the percentage ofinhibition (range: from zero to 100.0%), which contributes to thereliability of this in silico-in vitro trend.

To identify the molecular reasons for the two extremes of cellproliferation inhibition (highest and lowest percentage inhibitionfor (6b) and (3d), respectively), we performed a detailed analysis ofthe intermolecular interactions between the target (IKKb) and thedocked molecules. The superimposition of molecules (6b), (3d) andco-crystallized ligand (named “K252A”) can be seen in Fig. 10.

The difference between the binding modes of the (6b) and (3d)molecules is shown in Fig. 11 and Table 3. The residues of the IKKbtarget that participate in hydrophobic interactions, hydrogen bondsand pep interactions are highlighted in Fig. 11. It seems that theadditional hydrophobicity of the Cl-phenyl group in molecule (6b)provides a greater contact surface for interactions with hydropho-bic residues of the target in comparison with molecule (3d), whichensures more stability in the complex formed with IKKb.

This stability difference is also revealed when we analyse the

Fig. 9. Trends observed between the cell proliferation inhibition and in silico (docking Chemsquare for the HCT-8 tumour line and the green triangle for the MDA/MB-435 tumour line. (Fto the web version of this article.)

docking score values for the (6b) and (3d) molecules, which are78.07 and 62.15, respectively. Due to the trend observed betweenthe in silico docking scores (ChemPLP scores) and the in vitrocytotoxic activity on tumour cells, the molecules with high affinity(in silico) for the IKKb target seem to prevent more cell proliferation(in vitro), at least for the three tumour lines tested in this work (SF-295, HCT-8 and MDA/MB-435).

3. Conclusions

The current investigation has described the facile synthesis ofanti-cancer compounds, which showed significant cytotoxic activ-ity toward three human cancer cell lines and immunosuppressiveactivity over cytokines TNFa, IFNg, IL-2 and IL-6. In silico dockingstudies have shown that the molecules with more stable or positivedocking scores (i.e., greater in silico affinity for the IKKb target) arealso the most cytotoxic in human cancer cell lines. In summary,compounds (6b) and (6f) hold potential as immunosuppressiveagents with anticancer properties. The described findings mayopen up new possibilities for developing a new class of drugs withimmunosuppressive and cytotoxic activity.

4. Experimental methods

4.1. General

Melting points were measured with a Fisatom (Mod. 430D,60 Hz) melting point apparatus and are uncorrected. 1H NMR

PLP score) results. The blue rhombus shows trends for the SF-295 tumour line, the redor interpretation of the references to colour in this figure legend, the reader is referred

Fig. 10. Superimposition of the docking solutions for compounds (6b) (blue stick), (3d) (red stick) and the crystallographic structure of the “K252A” co-crystallized ligand (grayline). (A) Full view and (B) active site view. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 11. Detailed view of the docking solutions for (A) compound (6b) and (B) compound (3d). Residues involved in hydrophobic interactions (green), hydrogen bonds (cyan), andpep T-shaped interactions (orange) are highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 3Molecular interaction of IKKb with molecules (6b) and (3d).

Residues Molecules

6b 3d

LEU21 HC HCGLY22 e HCTHR23 e 3.0VAL29 HC eARG31 HC eALA42 HC HCLYS44 HC eVAL74 HC HCMET96 HC HCGLU97 2.9 eTYR98 HC PICYS99 3.3 HCASP103 e 3.3ILE165 HC e

Docking score 78.07 62.15

HC means hydrophobic contacts, PI means pep interaction, and the numbers arethe hydrogen bond distances between donor and acceptor, in Ångstroms. (SeeFig. 11).


spectra were recorded on a 300 MHz spectrometer in appropriatesolvents using TMS as an internal standard or the solvent signals assecondary standards. The chemical shifts are shown in d (ppm)scale. Multiplicities of NMR signals are designated as s (singlet),d (doublet), br (broad) and m (multiplet, for unresolved lines). 13CNMR spectra were recorded on a 75.5 MHz spectrometer. All theexperiments were monitored by analytical thin layer chromatog-raphy (TLC) performed on silica gel GF254 pre-coated plates. Afterelution, plates were visualized under UV illumination at 254 nm forUV active materials.

4.2. General procedure for the synthesis of thiazolidinones (3aed).Example for compound (3a)

Thiosemicarbazone (2a) (0.4 g, 1.52 mmol), anhydrous sodiumacetate (0.5 g, 6.08 mmol), and 50 mL ethanol were added to a100 mL round bottom flask under magnetic stirring and slightlywarmed for 10e15 min. Then, ethyl 2-bromoacetate (0.26 g,1.52 mmol) was added, and the colourless reaction was kept underreflux heating for 18 h. After cooling the solution back to roomtemperature (r.t.), the precipitate was filtered off and the solventwas evaporated for half of its volume and then cooled to 0 �C. Awhite solid was obtained, filtered in a Büchner funnel with a


sintered disc filter, washed with cold water and then dried in SiO2.Products were purified by recrystallization using the solvent sys-tem detailed below for each compound.

4.2.1. 2-(4-Oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (3a)

After crystallization with toluene, colourless crystals were ob-tained; yield¼ 74%; M.p. (�C): 262e263; IR (KBr) 3075 (NeH), 2968(CeH), 1771 and 1714 (C]O), 1641 (C]N) cm�1. 1H NMR (300 MHz,DMSO-d6): d 3.68 (s, 2H, CH2 heterocycle); 4.47 (d, 2H, CH2); 7.71 (t,1H, CH); 7.82e7.92 (m, 4H, Ar); 11.84 (s, 1H, NH). 13C NMR(75.5 MHz, DMSO-d6): d 32.8 (CH2 heterocycle); 37.6 (CH2); 123.1(CH Ar); 131.73 (CH Ar); 134.5 (Ar); 153.5 (HC]N); 165.7 (SeC]N);167.5 (C]O); 167.6 (C]O); 174.4 (C]O heterocycle). Anal. Calcd.For (3a): C, 51.65; H, 3.33; N, 18.53; S, 10.61; Found: C, 51.42; H,3.40; N, 18.13; S, 10.36. Rf: 0.35.

4.2.2. 2-(5-Methyl-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (3b)

After crystallization with toluene, colourless crystals were ob-tained; yield ¼ 70%; M.p. (�C): 227; IR (KBr) 3461 (NeH), 3034(CeH), 1772 and 1718 (C]O), 1648 (C]N) cm�1. 1H NMR (300MHz.DMSO-d6): d 1.35 (d, 3H, CH3); 4.00 (q, 1H, CH); 4.47 (d, 2H, CH2);7.71 (t, 1H, CH); 7.84e7.93 (m, 4H, Ar); 11.82 (s, 1H, NH). 13C NMR(75.5 MHz, DMSO-d6): d 18.7 (CH3); 37.4 (CH2); 42.1 (CH hetero-cycle); 123.2 (CH Ar); 131.7 (CH Ar); 134.6 (Ar); 153.8 (HC]N);163.9 (SeC]N); 167.5 (C]O); 177.4 (C]O heterocycle). Anal. Calcd.For (3b): C, 53.16; H, 3.82; N, 17.71; S, 10.14; Found: C, 53.55; H,3.90; N, 17.43; S, 10.08. Rf: 0.526.

4.2.3. 2-(5-Ethyl-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (3c)

After crystallization with toluene, colourless crystals were ob-tained; yield ¼ 79%; M.p. (�C): 191e192; IR (KBr) 3397 (NeH), 2963(CeH), 1770 and 1714 (C]O), 1651 (C]N) cm�1. 1H NMR (300 MHz,DMSO-d6): d 0.80 (t, 3H, CH3); 1.59e1.98 (m, 2H, CH2); 4.36 (d, 2H,CH2); 7.43 (t,1H, CH heterocycle); 7.68 (t, 1H, CH); 7.83e7.91 (m, 4H,Ar); 11.23 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): d 10.3 (CH3);25.4 (CH2); 37.6 (CH2); 49.3 (CH heterocycle); 123.0 (Ar); 131.5 (Ar);134.2 (Ar); 140.3 (HC]N); 152.6 (C]N); 167.3 (C]O); 177.2 (C]O);178.0 (C]O). Anal. Calcd. For (3c): C, 54.53; H, 4.27; N, 16.96; S,9.71; Found: C, 54.37; H, 4.42; N, 17.16; S, 10.05. Rf: 0.5.

4.2.4. 2-(3-Methyl-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (3d)

After crystallization with toluene, colourless crystals were ob-tained; yield ¼ 65%; M.p. (�C): 180e181; IR (KBr) 2979 (CeH), 1770and 1718 (C]O), 1637 (C]N) cm�1. 1H NMR (300 MHz. DMSO-d6):d 3.04 (s, 3H, CH3); 3.74 (s, 2H, CH2 heterocycle); 4.49 (d, 2H, CH2);7.40 (t, 1H, CH); 7.83e7.90 (m, 4H, Ar). 13C NMR (75.5 MHz. DMSO-d6): d 29.1 (CH2 heterocycle); 30.6 (CH2); 31.81 (CH3); 123.0 (CH Ar);131.6 (CH Ar); 134.4 (Ar); 154.8 (HC]N); 164.8 (SeC]N); 167.4(C]O); 172.1 (C]O); 178.0 (C]O heterocycle). Anal. Calcd. For(3d): C, 51.16; H, 3.82; N, 17.71; S, 10.14; Found: C, 51.40; H, 4.02; N,17.42; S, 9.89. Rf: 0.562.

4.3. General procedure for the synthesis of benzylidenes (4aef).Example for benzylidene (4a)

Thiazolidinone (3a) (0.4 g, 1.32 mmol), anhydrous potassiumacetate (0.39 g, 3.96 mmol), and 5 mL dimethylformamide wereadded to a 100 mL round bottom flask under magnetic stirring andslightly warmed for 10e15min. Then 4-fluorobenzaldehyde (0.16 g,1.32 mmol) was added and the reaction acquired yellow colour waskept under heating under reflux for 24 h. After cooling back to r.t.,

water was added to the flask and a yellow precipitate was formed.The precipitate was filtered off and the solvent was discarded. Ayellow solid is obtained, filtered in Büchner funnel with sintereddisc filter, washed with cold water, and then dried in SiO2. Productsare purified by column chromatography using the solvent systemdetailed below for each compound.

4.3.1. 2-(2-((-5-(4-Fluorobenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4a)

After elution with hexane/acetate (8:2), yellow crystals wereobtained; yield ¼ 38%; M.p. (�C): unidentified to 300 �C; IR (KBr)3471 (NeH), 2935 (CeH), 1716 (C]O), 1634 and 1600 (C]N), 1233(CeF) cm�1. 1H NMR (300 MHz, DMSO-d6): d 4.32 (d, 2H, CH2); 7.22(d, 2H, Ar); 7.48 (t, 1H, CH); 7.69 (s, 1H, CH); 7.72 (d, 2H, Ar);7.79e7.96 (m, 4H, Ar); 8.52 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): d 37.6 (CH2); 115.1 (SeC]C); 120.4 (CH Ar); 130.8 (CH Ar);123.6 (CH Ar); 131.8 (CH Ar); 132.2 (Ar); 132.7 (Ar); 142 (HC]C);162.1 (CeF); 163 (C]N); 163.7 (HC]N); 168.2 (C]O); 170.2 (C]O).Anal. Calcd. For (4a): C, 58.82; H, 3.21; N, 13.72; S, 7.85; Found: C,58.74; H, 3.48; N, 13.81; S, 8.03. Rf: 0.631.

4.3.2. 2-(2-((-5-(4-Chlorobenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4b)

After elution with hexane/acetate (8:2), yellow crystals wereobtained; yield ¼ 46%; M.p. (�C): unidentified to 300 �C; IR (KBr)2925 (CeH), 1724 (C]O), 1596 (C]N), 821 (CeCl) cm�1. 1H NMR(300 MHz, DMSO-d6): d 4.35 (d, 2H, CH2); 7.55 (t, 1H, CH); 7.60 (d,2H, Ar); 7.63 (s, 1H, CH); 7.67 (d, 2H, Ar); 7.82e7.99 (m, 4H, Ar); 8.54(s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): d 37.6 (CH2); 116.0(SeC]C); 123.3 (CH Ar); 129.5 (CH Ar); 129.7 (CH Ar); 131.9 (CHAr); 133.3 (Ar); 133.5 (Ar); 134.7 (Ar); 142 (HC]C); 163.7 (HC]N);165.0 (C]N); 168.2 (C]O); 170.0 (C]O); 170.2 (C]O). Anal. Calcd.For (4b): C, 56.54; H, 3.08; N,13.19; S, 7.55; Found: C, 56.37; H, 3.07;N, 13.49; S, 7.77. Rf: 0.635.

4.3.3. 2-(2-((-5-(4-Bromobenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4c)

After elution with hexane/acetate (8:2), yellow crystals wereobtained; yield ¼ 60%; M.p. (�C): unidentified to 300 �C; IR (KBr)2933 (CeH), 1716 (C]O), 1607 (C]N), 558 (CeBr) cm�1. 1H NMR(300 MHz, DMSO-d6): d 4.37 (d, 2H, CH2); 7.50 (t, 1H, CH); 7.55 (d,2H, Ar); 7.64 (d, 2H, Ar); 7.72 (s,1H, CH); 7.80e8.06 (m, 4H, Ar); 8.55(s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): d 37.6 (CH2); 116.3(SeC]C); 122.3 (CH Ar); 122.9 (CH Ar); 128.6 (CH Ar); 131.5 (CHAr); 132.3 (Ar); 132.7 (Ar); 134.2 (Ar); 142 (HC]C); 163.7 (HC]N);164 (C]N); 168.4 (C]O); 173.1 (C]O). Anal. Calcd. For (4c): C,51.18; H, 2.79; N, 11.94; S, 6.83; Found: C, 50.99; H, 2.76; N, 11.80; S,7.11. Rf: 0.625.

4.3.4. 2-(2-((-5-Benzylidene-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4d)

After elution with hexane/acetate (8:2), yellow crystals wereobtained; yield ¼ 56%; M.p. (�C): unidentified to 300 �C; IR (KBr)3026 (CeH), 1715 (C]O), 1608 (C]N) cm�1. 1H NMR (300 MHz,DMSO-d6): d 3.95 (d, 2H, CH2); 7.53 (t, 1H, CH); 7.41e7.89 (m, 10H;9H Ar and 1H CH); 8.50 (NH). 13C NMR (75.5 MHz, DMSO-d6): d 40.3(CH2); 116.0 (SeC]C); 122.9 (CH Ar); 127.3 (CH Ar); 127.9 (CH Ar);128.8 (CH Ar); 132.3 (CH Ar); 133.7 (Ar); 135.2 (Ar); 142.5 (HC]C);158.4 (HC]N); 159.9 (C]N); 164.6 (C]O); 168 (C]O). Anal. Calcd.For (4d): C, 61.53; H, 3.61; N, 14.35; S, 8.21; Found: C, 61.47; H, 3.77;N, 14.77; S, 8.49. Rf: 0.66.

4.3.5. 2-(2-((-5-(3-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4e)

After elution with hexane/acetate (8:2), yellow crystals were


obtained; yield ¼ 57%; M.p. (�C): unidentified to 300 �C; IR (KBr)3402 (NeH), 2952 (CeH), 1718 (C]O), 1642 and 1594 (C]N), 1265(CeO) cm�1. 1H NMR (300 MHz, DMSO-d6): d 3.80 (s, 1H, CH3); 4.53(d, 2H, CH2); 6.89e7.85 (m, 10H; 8H Ar and 2H CH); 8.55 (NH). 13CNMR (75.5 MHz, DMSO-d6): d 43.1 (CH2); 56.8 (CH3); 113.5 (CH Ar);116.0 (SeC]C); 120.9 (CH Ar); 124.2 (CH Ar); 127.9 (CH Ar); 130.8(Ar); 134.0 (Ar); 136.2 (HC]C); 158.3 (HC]N); 159.9 (CeO Ar);168.0 (C]O); 168.2 (C]O). Anal. Calcd. For (4e): C, 59.99; H, 3.84;N, 13.33; S, 7.63; Found: C, 59.56; H, 3.78; N, 12.94; S, 7.28. Rf: 0.58.

4.3.6. 2-(2-((-5-(4-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4f)

After elution with hexane/acetate (8:2), yellow crystals wereobtained; yield ¼ 35%; M.p. (�C): unidentified to 300 �C; IR (KBr)2932 (CeH), 1717 (C]O), 1597 (C]N), 1256 (CeO) cm�1. 1H NMR(300 MHz, DMSO-d6): d 3.85 (s, 1H, CH3); 4.56 (d, 2H, CH2); 6.99 (d,2H, Ar); 7.51 (t, 1H, CH); 7.63 (d, 2H, Ar); 7.73 (s, 1H, CH); 7.83e7.88(m, 4H, Ar); 8.55 (NH). 13C NMR (75.5 MHz, DMSO-d6): d 40.1 (CH2);57.1 (CH3); 114.6 (CH Ar); 116.0 (SeC]C); 123.3 (CH Ar); 128.4 (CHAr); 130.7 (CH Ar); 132.0 (Ar); 142.0 (HC]C); 157.9 (CeO Ar); 159.7(HC]N); 160 (C]N); 163.3 (C]O); 168.2 (C]O). Anal. Calcd. For(4f): C, 59.99; H, 3.84; N, 13.33; S, 7.63; Found: C, 59.67; H, 3.62; N,13.47; S, 7.57. Rf: 0.632.

4.4. General procedure for the synthesis of benzylidenes (6aef).Example for benzylidene (6a)

Thiazolidinone (5) (0.4 g, 1.52 mmol), anhydrous potassiumacetate (0.45 g, 4.56 mmol), and 5 mL of dimethylformamide wereadded to a 100 mL round bottom flask under magnetic stirring andslightly warmed for 10e15min. Next, 4-fluorobenzaldehyde (0.19 g,1.52 mmol) was added, and the reaction acquired a yellow colourand was kept under heating under reflux for 24 h. After coolingback to r.t., water was added to the flask and a yellow precipitatewas formed. The precipitate was filtered off and the solvent wasdiscarded. A yellow solid was obtained, filtered in Büchner funnelwith a sintered disc filter, washed with cold water, and then driedin SiO2. Products were purified by column chromatography usingthe solvent system detailed below for each compound.

4.4.1. 2-((-5-(4-Fluorobenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6a)

After elution with hexane/acetate (6:4), yellow crystals wereobtained; yield ¼ 36%; M.p. (�C): unidentified to 300 �C; IR (KBr)2950 (CeH), 1709 (C]O), 1651 and 1598 (C]N), 1231 (CeF) cm�1.1H NMR (300 MHz, DMSO-d6): d 7.21 (d, 2H, Ar); 7.61 (d, 2H, Ar);7.72 (s, 1H, CH); 7.84e7.88 (m, 4H, Ar); 8.40 (s, 1H, NH). 13C NMR(75.5 MHz, DMSO-d6): d 115.4 (CH Ar); 116.0 (SeC]C); 122.8 (CHAr); 124.9 (CH Ar); 127.4 (CH Ar); 131.6 (Ar); 132.2 (Ar); 133.0 (HC]C); 141.6 (C]N); 144.7 (CeF Ar); 167.7 (C]O); 169.8 (C]O); 175.1(C]O). Anal. Calcd. For (6a): C, 58.85; H, 2.74; N, 11.44; S, 8.73;Found: C, 58.83; H, 2.58; N, 11.51; S, 8.56. Rf: 0.697.

4.4.2. 2-((-5-(4-Chlorobenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6b)

After elution with hexane/acetate (6:4), yellow crystals wereobtained; yield ¼ 51%; M.p. (�C): unidentified to 300 �C; IR (KBr)2938 (CeH), 1708 (C]O), 1647 (C]N), 745 (CeCl) cm�1. 1H NMR(300 MHz, DMSO-d6): d 7.40 (d, 2H, Ar); 7.62 (d, 2H, Ar); 7.71 (s, 1H,CH); 7.85e7.89 (m, 4H, Ar); 8.57 (s, 1H, NH). 13C NMR (75.5 MHz,DMSO-d6): d 115.0 (SeC]C); 125.3 (CH Ar); 128.8 (CH Ar); 129.0(CH Ar); 131.9 (CH Ar). 132.2 (Ar); 133.5 (Ar); 133.7 (CeCl Ar); 140.5(HC]C); 144.1 (C]N); 159.8 (C]O); 161.3 (C]O). Anal. Calcd. For(6b): C, 56.33; H, 2.63; N, 10.95; S, 8.35; Found: C, 56.20; H, 2.32; N,10.96; S, 8.17. Rf: 0.526.

4.4.3. 2-((-5-(4-Bromobenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6c)

After elution with hexane/acetate (6:4), yellow crystals wereobtained; yield ¼ 33%; M.p. (�C): unidentified to 300 �C; IR (KBr)3434 (NH), 2937 (CeH), 1715 (C]O), 1645 and 1583 (C]N), 558(CeBr) cm�1. 1H NMR (300 MHz, DMSO-d6): d 7.53 (d, 2H, Ar); 7.61(d, 2H, Ar); 7.73 (s, 1H, CH); 7.86e7.90 (m, 4H, Ar); 8.59 (s, 1H, NH).13C NMR (75.5 MHz, DMSO-d6): d 116.0 (SeC]C); 122.5 (CH Ar);128.7 (CH Ar); 124.0 (CH Ar). 131.3 (CH Ar); 132.0 (Ar); 132.3 (CHAr); 142.0 (HC]C); 144.6 (C]N); 159.9 (C]O); 165.4 (C]O). Anal.Calcd. For (6c): C, 50.48; H, 2.35; N, 9.81; S, 7.49; Found: C, 50.22; H,2.58; N, 9.62; S, 7.20. Rf: 0.552.

4.4.4. 2-((-5-Benzylidene-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6d)

After elution with hexane/acetate (6:4), yellow crystals wereobtained; yield ¼ 40%; M.p. (�C): unidentified to 300 �C; IR (KBr)3023 and 2951 (CeH), 1712 (C]O), 1646 and 1593 (C]N) cm�1. 1HNMR (300 MHz, DMSO-d6): d 7.45e7.85 (m, 9H, Ar); 8.40 (s, 1H,CH); 8.51 (s, 1H, NH). 13C NMR (75.5MHz, DMSO-d6): d 115.6 (SeC]C); 128.6 (CH Ar); 128.8 (CH Ar); 129.8 (CH Ar); 129.9 (CH Ar); 130.2(CH Ar); 130.6 (Ar); 130.8 (Ar); 131.8 (HC]C); 134.8 (C]N); 157.2(C]O); 158.0 (C]O). Anal. Calcd. For (6d): C, 61.88; H, 3.17; N,12.03; S, 9.18; Found: C, 61.50; H, 3,28; N, 12.39; S, 9.46. Rf: 0.592.

4.4.5. 2-((-5-(3-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6e)

After elution with hexane/acetate (6:4), yellow crystals wereobtained; yield ¼ 22%; M.p. (�C): unidentified to 300 �C; IR (KBr)3460 (NeH), 2936 (CeH), 1711 (C]O), 1641 and 1594 (C]N), 1266(CeO) cm�1. 1H NMR (300 MHz, DMSO-d6): d 3.99 (s, 3H, CH3);6.89e7.46 (m, 4H, Ar). 7.72 (s, 1H, CH); 7.85e7.88 (m, 4H, Ar); 8.51(NH). 13C NMR (75.5 MHz, DMSO-d6): d 57.2 (CH3); 114.3 (CH Ar);116.0 (SeC]C); 120.1 (CH Ar); 122.6 (CH Ar); 128.2 (CH Ar); 132.5(CH Ar). 132.8 (Ar); 136.8 (Ar); 139.1 (HC]C); 142.0 (C]N); 157.4(CeOAr); 159.2 (C]O); 163.6 (C]O). Anal. Calcd. For (6e): C, 60.15;H, 3.45; N, 11.08; S, 8.45; Found: C, 60.01; H, 3.25; N, 11.20; S, 8.47.Rf: 0.578.

4.4.6. 2-((-5-(4-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6f)

After elution with hexane/acetate (6:4), yellow crystals wereobtained; yield ¼ 46%; M.p. (�C): unidentified to 300 �C; IR (KBr)2952 (CeH), 1710 (C]O), 1651 and 1598 (C]N), 1255 (CeO) cm�1.1H NMR (300 MHz, DMSO-d6): d 4.01 (s, 3H, CH3); 6.69 (d, 2H, Ar);7.65 (d, 2H, Ar); 7.84e7.89 (m, 4H, Ar); 8.38 (s, 1H, CH); 8.49 (s, 1H,NH). 13C NMR (75.5 MHz, DMSO-d6): d 55.9 (CH3); 115.2 (CH Ar);116.0 (SeC]C); 124.4 (CH Ar); 128.3 (CH Ar); 132.1 (CH Ar); 133.6(Ar); 133.9 (Ar); 142.8 (HC]C); 144.3 (C]N); 148.3 (CeOAr); 160.3(C]O); 163.2 (C]O). Anal. Calcd. For (6f): C, 60.15; H, 3.45; N,11.08;S, 8.45; Found: C, 60.48; H, 3.74; N, 11.05; S, 8.23. Rf: 0.605.

4.5. General procedure for the synthesis of 1,3-thiazoles (7aeh).Example for thiazole (7a)

Thiosemicarbazone (2a) (0.15 g, 0.57 mmol), anhydrous sodiumacetate (0.18 g, 2.28 mmol), and 50 mL ethanol were added to a100 mL round bottom flask under magnetic stirring and slightlywarmed for 10e15 min. Then, 2-bromoacetophenone (0.11 g,0.57mmol) was added, and the reaction acquired purple colour andwas kept under heating under reflux for 4 h. After cooling back tor.t., the precipitate was filtered off and the solvent was evaporatedfor half of its volume and then cooled to 0 �C. A purple solid wasobtained, filtered in Büchner funnel with a sintered disc filter,washed with cold water, and then dried in SiO2. Products were


purified by recrystallization using the solvent system detailedbelow for each compound.

4.5.1. 2-(4-Phenylthiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7a)

After crystallization with water, purple crystals were obtained;yield ¼ 59%; M.p. (�C): 207e209; IR (KBr) 3262 (NeH), 3039(CeH), 1767 and 1712 (C]O), 1561 (C]N) cm�1. 1H NMR(300 MHz, DMSO-d6): d 4.44 (d, 2H, CH2); 7.16 (s, 1H, CH hetero-cycle); 7.26 (t, 1H, CH); 7.36 (t, 2H, Ar, 1H, CeH); 7.77 (d, 2H, Ar);7.91 (m, 4H, Ar); 11.88 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):d 37.4 (CH2); 104.4 (SeCH heterocycle); 124.2 (CH Ar); 126.4 (CHAr); 128.5 (CH Ar); 129.6 (CH Ar); 132.7 (CH Ar); 135.6 (Ar); 139.1(HC]N); 151.3 (C]N); 168.6 (C]O); 169.2 (C]O). Anal. Calcd. For(7a): C, 62.97; H, 3.89; N, 15.46; S, 8.85; Found: C, 62.69; H, 4.19; N,15.68; S, 8.98. Rf: 0.625.

4.5.2. 2-(4-Methylthiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7b)

After crystallization with toluene, brown crystals were ob-tained; yield ¼ 55%; M.p. (�C): 209e210; IR (KBr) 3410 (NeH), 2934(CeH), 1772 and 1715 (C]O), 1639 (C]N) cm�1. 1H NMR (300 MHz,DMSO-d6): d 2.08 (s, 3H, CH3); 4.39 (d, 2H, CH2); 6.20 (s, 1H, CHheterocycle); 7.30 (t, 1H, CH); 7.84e7.92 (m, 4H, Ar); 11.51 (s, 1H,NH). 13C NMR (75.5 MHz, DMSO-d6): d 18.1 (CH3); 37.4 (CH2); 103.0(SeCH heterocycle); 124.1 (CH Ar); 132.7 (CH Ar); 135.5 (Ar); 138.9(NeC]C); 148.3 (HC]N); 155.0 (C]N); 168.6 (C]O); 168.9 (C]O). Anal. Calcd. For (7b): C, 55.99; H, 4.03; N, 18.65; S, 10.68; Found:C, 55.76; H, 4.37; N, 18.48; S, 10.87. Rf: 0.3.

4.5.3. 2-(4-(4-Fluorophenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7c)

After crystallization with water, white crystals were obtained;yield ¼ 69%; M.p. (�C): 214e126; IR (KBr) 3460 (NeH), 3102 (CeH),1772 and 1718 (C]O), 1571 (C]N), 1394 (CeF) cm�1. 1H NMR(300 MHz, DMSO-d6): d 4.42 (d, 2H, CH2); 7.12 (s, 1H, CH hetero-cycle); 7.35 (t,1H, CH); 7.77e7.82 (m, 4H, Ar); 7.85e7.94 (m, 4H, Ar);11.88 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): d 37.4 (CH2); 104.1(SeCH heterocycle); 116.6 (CH Ar); 124.2 (CH Ar); 128.4 (CH Ar);132.3 (CH Ar); 132.7 (Ar); 135.6 (Ar); 139.3 (CeF Ar); 150.2 (NeC]C); 150.3 (HC]N); 160.9 (C]N); 168.7 (C]O); 169.4 (C]O). Anal.Calcd. For (7c): C, 59.99; H, 3.44; N, 14.73; S, 8.43; Found: C, 60.08;H, 3.61; N, 15.01; S, 8.24. Rf: 0.625.

4.5.4. 2-(4-(4-Nitrophenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7d)

After crystallization with water, yellow crystals were obtained;yield ¼ 72%; M.p. (�C): 240; IR (KBr) 3229 (NeH), 3110 (CeH), 1768and 1705 (C]O), 1572 (C]N), 1510 and 1345 (NO2) cm�1. 1H NMR(300 MHz, DMSO-d6): d 4.42 (d, 2H, CH2); 7.39 (t, 1H, CH); 7.57 (s,1H, CH); 7.86e7.98 (m, 4H, Ar); 8.05 (d, 2H, Ar); 8.22 (d, 2H, Ar);11.96 (s, 1H, NH). 13C NMR (75.5MHz, DMSO-d6): d 37.4 (CH2); 108.3(CH heterocycle); 123.2 (CH Ar); 124.1 (CH Ar); 126.3 (CH Ar); 131.7(CH Ar); 134.6 (Ar); 140.6 (Ar); 146.1 (Ar); 150.2 (NeC]C); 154.7(HC]N); 160.9 (C]N); 167.6 (C]O); 168.8 (C]O). Anal. Calcd. For(7d): C, 56.01; H, 3.22; N, 17.19; S, 7.87; Found: C, 55.78; H, 3.27; N,16.95; S, 8.15. Rf: 0.71.

4.5.5. 2-(4-(4-Methoxyphenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7e)

After crystallization with water, white crystals were obtained;yield ¼ 84%; M.p. (�C): 225; IR (KBr) 3273 (NeH), 3115 (CeH), 1766and 1711 (C]O), 1563 (C]N), 1248 (CeO) cm�1. 1H NMR (300 MHz,DMSO-d6): d 3.7 (d, 2H, CH2); 3.85 (s, 3H, CH3); 7.05 (d, 2H, Ar); 7.28(s, 1H, CH heterocycle); 7.50 (t, 1H, CH2); 7.55 (d, 2H, Ar); 7.85e7.88

(m, 4H, CH Ar); 11.99 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):d 37.4 (CH2); 55.1 (CH3); 101.1 (CH heterocycle); 113.9 (CH Ar); 123.1(CH Ar); 126.7 (CH Ar); 127.5 (CH Ar); 131.7 (Ar); 134.5 (Ar); 138.0(Ar); 150.2 (NeC]C); 154.1 (HC]N); 158.7 (C]N); 167.6 (C]O);168.1 (C]O). Anal. Calcd. For (7e): C, 61.21; H, 4.11; N, 14.28; S, 8.17;Found: C, 61.03; H, 4.28; N, 13.91; S, 7.87. Rf: 0.63.

4.5.6. 2-(4-(4-Bromophenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7f)

After crystallization with water, white crystals were obtained;yield ¼ 72%; M.p. (�C): 190; IR (KBr) 3471 and 3403 (NeH), 2955(CeH), 1770 and 1716 (C]O), 1560 (C]N), 719 (CeBr) cm�1. 1HNMR (300 MHz, DMSO-d6): d 4.40 (d, 2H, CH2); 7.28 (s, 1H, CHheterocycle); 7.31 (t, 1H, CH); 7.49 (d, 2H, Ar); 7.57 (d, 2H, Ar);7.85e7.94 (m, 4H, Ar); 11.73 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): d 37.4 (CH2); 105.0 (CH heterocycle); 120.0 (CeBr); 123.2 (CHAr); 129.7 (CH Ar); 131.2 (CH Ar); 131.7 (CH Ar); 134.3 (Ar); 134.5(Ar); 138.5 (NeC]C); 155.7 (HC]N); 164.4 (C]N); 167.6 (C]O).Anal. Calcd. For (7f): C, 51.71; H, 2.97; N, 12.70; S, 7.27; Found: C,51.42; H, 3.13; N, 12.93; S, 7.17. Rf: 0.73.

4.5.7. 2-(4-(4-Chlorophenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7g)

After crystallization with water, white crystals were obtained;yield ¼ 73%; M.p. (�C): 216; IR (KBr) 3465 (NeH), 3049 (CeH), 1774and 1719 (C]O), 1550 (C]N), 720 (CeCl) cm�1. 1H NMR (300 MHz,DMSO-d6): d 4.25 (d, 2H, CH2); 7.28 (CH heterocycle); 7.32 (d, 2H,Ar); 7.44 (d, 2H, Ar); 7.53 (t, 1H, CH); 7.84e7.95 (m, 4H, Ar); 11.85 (s,1H, NH). 13C NMR (75.5 MHz, DMSO-d6): d 37.4 (CH2); 104.5 (CHheterocycle); 123.1 (CH Ar); 127.1 (CH Ar); 128.5 (CH Ar); 131.6 (CHAr); 131.8 (Ar); 134.5 (Ar); 138.3 (Ar); 150.2 (NeC]C); 155.7 (HC]N); 159.3 (C]N); 167.5 (C]O); 168.2 (C]O). Anal. Calcd. For (7g):C, 57.50; H, 3.30; N, 14.12; S, 8.08; Found: C, 57.41; H, 3.44; N, 14.02;S, 7.88. Rf: 0.75.

4.5.8. 2-(4-p-Tolylthiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7h)

After crystallization with water, white crystals were obtained;yield ¼ 53%; M.p. (�C): 209e210; IR (KBr) 3281 (NeH), 3029 (CeH),1775 and 1717 (C]O), 1552 (C]N) cm�1. 1H NMR (300 MHz,DMSO-d6): d 2.34 (s, 3H, CH3); 4.13 (d, 2H, CH2); 6.98 (s, 1H, CHheterocycle); 7.38 (t, 1H, CH); 7.52 (d, 2H, Ar); 7.65 (d, 2H, Ar);7.87e7.92 (m, 4H, Ar); 11.80 (s, 1H, NH). 13C NMR (75.5MHz, DMSO-d6): d 21.3 (CH3); 37.4 (CH2); 102.8 (CH heterocycle); 123.5 (CH Ar);125.8 (CH Ar); 129.5 (CH Ar); 132.1 (CH Ar); 132.4 (Ar); 134.9 (Ar);137.1 (Ar); 138.5 (NeC]C); 154.7 (HC]N); 151.8 (C]N); 168.0 (C]O); 168.6 (C]O). Anal. Calcd. For (7h): C, 63.81; H, 4.28; N, 14.88; S,8.52; Found: C, 63.77; H, 4.26; N, 14.69; S, 8.29. Rf: 0.684.

4.6. Molecular modelling

The structures of all compounds were obtained by applying theRM1 method [55], which is available as part of the SPARTAN'08program [56] by using internal default settings for convergencecriteria. Docking calculations and analyses were carried out usingthe structure of human IkB Kinase b e IKKb e (PDB ID code: 4KIK)as the target, which is composed of a co-crystallized complex withthe inhibitor, referred as “K252A” [57]. The active site was definedas all atoms within a radius of 6.0 Å from the co-crystallized ligand.The residues LEU21, THR23, LYS44, MET96, GLU97, TYR98, CYS99,ASP103, ILE165 and ASP166 were treated as flexible during thecalculations. The GOLD 5.2 program [58] was used for dockingcalculations. Next, the Binana program [59] was used to analyse themolecular interactions present in the best docking solutions, usingdefault settings except for the hydrogen bond distance, which was


changed to a maximum of 3.5 Å. Figures were generated withPymol [60].

4.7. Biological in vitro evaluation

4.7.1. Measuring cytotoxicity against tumour cell lines by MTT assayThe cytotoxicity of the compounds was evaluated by MTT assay

against three human cancer cell lines: SF-295 (nervous system),HCT-8, (colon) and MDA/MB-435 (melanoma), all of which wereobtained from the National Cancer Institute (NCI, Bethesda, MD,USA). Cell lines were maintained in RPMI 1640 medium supple-mented with 10% foetal bovine serum, 2 mM glutamine, 100 U/mLpenicillin and 100 mg/mL streptomycin, at 37 �C with 5% CO2.Tumour cell proliferation was quantified indirectly through theability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to form apurple formazan product [61]. Briefly, cells were plated in 96-wellplates and the compounds (50 mg/mL) were added to wells. After69 h of incubation, the supernatant was replaced with fresh me-dium containing 10% MTT. Three hours later, the MTT formazanproduct was dissolved in 150 mL DMSO, and the absorbance wasmeasured at 595 nm (DTX-880, Beckman Coulter). Doxorubicin(Dox, Sigma Aldrich) was used as a positive control (0.3 mg/mL). Toavoid false proliferation data, the experiments were performed intriplicate and the proliferation rate was always compared tonegative controls. All replicates had similar inhibition rates, and cellproliferation rates in negative control wells were higher than thosein the treated wells.

4.7.2. AnimalsMale 4- to 6-week-old BALB/c mice were used. All mice were

raised and maintained at the animal facilities of the Gonçalo MonizResearch Centre, Fundaç~ao Oswaldo Cruz, Salvador, Brazil, in roomswith controlled temperature (22 ± 2 �C), humidity (55 ± 10%) andcontinuous air renovation. Animals were housed in a 12 h light/12 hdark cycle (6 ame6 pm) and provided with rodent diet and waterad libitum. This study had prior approval by the Institutional EthicsCommittee in Laboratory Animal Use.

4.7.3. Macrophage cell culturesPeritoneal cells were obtained by washing, with cold Dulbecco's

modified Eagle's medium (DMEM; Life Technologies, GIBCO-BRL,Gaithersburg, MD), the peritoneal cavity of mice 4e5 days afterinjection of 3% thioglycolate in saline (1.5 mL per mouse). Cellswere washed twice with DMEM, resuspended in DMEM supple-mented with 10% foetal bovine serum (Cultilab, Campinas, Brazil)and 50 mg/mL of gentamycin (Novafarma, An�apolis, Brazil), andplated in 96-well tissue culture plates at 2� 105 cells per 0.2mL perwell. After 2 h of incubation at 37 �C, non-adherent cells wereremoved by two washes with DMEM. Macrophages were thentreated with LPS (500 ng/mL) in the absence or presence of thecompounds at 1 and 10 mg/mL. Thalidomide and dexamethasonewere used as reference drugs. Cell supernatants were collected at4 h of incubation to determine TNF-a levels or at 24 h of incubationto determine IL-6 levels.

4.7.4. Lymphocyte cell cultureSpleen cells (105 cells/well) obtained from BALB/c mice were

added to 96-well plates containing DMEM supplemented with 10%foetal bovine serum (Cultilab) and 50 mg/mL of gentamycin(Novafarma). Cells were stimulated with 1 mg/mL of concanavalin A(Sigma) and treated with 1 and 10 mg/mL of the compounds, in afinal volume of 0.2 mL. Thalidomide and dexamethasonewere usedas reference drugs. Cell supernatants were collected at 24 h of in-cubation to determine IFN- g and IL-2 levels.

4.7.5. Cytokine determinationsCytokine concentrations were determined in cell-free culture

supernatants using specific sandwich ELISA kits for each cytokine,following the manufacturer's instructions (Duoset, R&D Systems,Minneapolis, MN, EUA).

Acknowledgements

We would like to thank the Brazilian National Research Council(CNPq) and the Research Foundation of Pernambuco State (FACEPE)for financial support. M.V.O.C. is recipient of a FACEPE scholarship(BFP-0107-4.03/12). A.C.L.L. is recipient of a CNPq fellowship(308806/2013-1). C.P. is grateful to FUNCAP (Fundaç~ao Cearense deApoio ao Desenvolvimento Científico de Tecnol�ogico). P.M.P.F. isalso grateful to FAPEPI (Fundaç~ao de Amparo �a Pesquisa do Estadodo Piauí) for financial support. Our thanks are also due to theDepartment of Chemistry at the Federal University of Pernambuco(UFPE) for recording the NMR (1H and 13C), IR spectra and theelemental analysis of all compounds.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2015.04.041.

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Design, synthesis and structure–activity relationship of phthalimides endowed with dual antiproliferative and immunomodulat ...1. Introduction2. Results and discussion2.1. Chemistry2.2. X-ray analysis2.3. Pharmacological evaluation2.4. Docking studies

3. Conclusions4. Experimental methods4.1. General4.2. General procedure for the synthesis of thiazolidinones (3a–d). Example for compound (3a)4.2.1. 2-(4-Oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (3a)4.2.2. 2-(5-Methyl-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (3b)4.2.3. 2-(5-Ethyl-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (3c)4.2.4. 2-(3-Methyl-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (3d)

4.3. General procedure for the synthesis of benzylidenes (4a–f). Example for benzylidene (4a)4.3.1. 2-(2-((-5-(4-Fluorobenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4a)4.3.2. 2-(2-((-5-(4-Chlorobenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4b)4.3.3. 2-(2-((-5-(4-Bromobenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4c)4.3.4. 2-(2-((-5-Benzylidene-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4d)4.3.5. 2-(2-((-5-(3-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4e)4.3.6. 2-(2-((-5-(4-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4f)

4.4. General procedure for the synthesis of benzylidenes (6a–f). Example for benzylidene (6a)4.4.1. 2-((-5-(4-Fluorobenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6a)4.4.2. 2-((-5-(4-Chlorobenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6b)4.4.3. 2-((-5-(4-Bromobenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6c)4.4.4. 2-((-5-Benzylidene-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6d)4.4.5. 2-((-5-(3-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6e)4.4.6. 2-((-5-(4-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)amino)isoindoline-1,3-dione (6f)

4.5. General procedure for the synthesis of 1,3-thiazoles (7a–h). Example for thiazole (7a)4.5.1. 2-(4-Phenylthiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7a)4.5.2. 2-(4-Methylthiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7b)4.5.3. 2-(4-(4-Fluorophenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7c)4.5.4. 2-(4-(4-Nitrophenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7d)4.5.5. 2-(4-(4-Methoxyphenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7e)4.5.6. 2-(4-(4-Bromophenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7f)4.5.7. 2-(4-(4-Chlorophenyl)thiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7g)4.5.8. 2-(4-p-Tolylthiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-dione (7h)

4.6. Molecular modelling4.7. Biological in vitro evaluation4.7.1. Measuring cytotoxicity against tumour cell lines by MTT assay4.7.2. Animals4.7.3. Macrophage cell cultures4.7.4. Lymphocyte cell culture4.7.5. Cytokine determinations

AcknowledgementsAppendix A. Supplementary dataReferences

European Journal of Medicinal Chemistry · 2018. 8. 15. · X-ray diffraction data collections were performed on an Enraf-Nonius Kappa-CCD diffractometer (95 mm CCD camera on k-goniostat)

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